PPARγ/PGC-1α Pathway in E-Cadherin Expression and Motility of HepG2 Cells

Materials and Methods

Cell culture. HepG2 human hepatoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 10 μg/ml gentamycin, 2 mM L-glutamine, 0.1 mM non-essential amino acid (complete DMEM) at 37°C in a humidified atmosphere containing 5% CO₂. All culture reagents were purchased from Invitrogen (Carlsbad, CA, USA); rosiglitazone and the specific PPARγ antagonist GW9662 were purchased from Cayman Chemical (Ann Arbor, MI, USA); troglitazone was purchased from Sigma-Aldrich (Saint Louis, MO, USA).

siRNA transfection. On-TARGETplus smart pools of small-interference (si)RNAs for non-targeting control, PPARγ (NM_005037) and E-cadherin (NM_004360) were synthesized by Dharmacon Research (Lafayette, CO, USA). Transient transfection was carried out using INTERFERin™ siRNA transfection reagent (Polyplus Transfection, New York, NY, USA) according to the manufacturer's protocol. Briefly, 0.1 nmol siRNAs and 12.5 μl INTERFERin™ were mixed thoroughly with 200 μl serum-free DMEM by vortexing for 10 s. After 10 min incubation at room temperature, this mixture was added to 2×10⁵ cells plated in 6-well plates containing 2 ml of complete DMEM. Two days post-transfection, cells were either collected or further infected with adenoviruses.

Ad-PGC-1α virus production and infection. Mass production and storage of Ad-PGC-1α and Ad-GFP viruses were carried out as described previously (19). For infection, HepG2 cells were incubated with the viruses in serum-free DMEM for two hours before virus-containing medium was replaced by fresh complete DMEM containing vehicle (0.1% dimethylformamide (DMF)) or PPARγ ligands (5 μM rosiglitazone, 20 μM troglitazone and 10 μM GW9662). After incubation for 2 days, cells were collected for RT-PCR or Western blot analysis of E-cadherin expression or for transwell migration assay.

Western blot analysis. Whole cell lysates were prepared by M-PER® (mammalian protein extraction reagent) and nuclear extracts were prepared by NE-PER® (nuclear and cytoplasmic extraction reagent) (Pierce, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA) according to the manufacturer's protocol. Protein lysates were separated in 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Primary antibodies, including those for E-cadherin (BD Biosciences, San Diego, CA, USA), β-actin (Sigma), PGC-1α, PPARγ and histone H1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), were then used to probe these blots. After incubation at 4°C overnight, the membranes were washed and then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein visualization was carried out using an enhanced chemiluminescence kit (ECL, Pierce). The signal intensity was obtained by densitometric scanning and relative protein expression was normalized against protein loading control (β-actin).

Sulforhodamine B (SRB) colorimetric analysis. HepG2 cells were seeded at a density of 3,000 cells/well in 96-well plates and were treated with different doses (0, 1, 5, 10, 20, 50 and 100 μM) of PPARγ agonists (rosiglitazone and troglitazone) for 2 days. The total biomass of cells was determined by SRB colorimetric assay (20). Briefly, cells were fixed by cold 10% trichloroacetic acid (TCA; Sigma) at 4°C for 1 hour. After washing with tap water and air drying, fixed cells were incubated with 0.1% SRB (Sigma) dissolved in 1% acetic acid for 30 min and rinsed five times with 1% acetic acid to remove unincorporated dye. The protein-bound dye was then extracted with 10 mM Tris and the absorbance at 510 nm of this extract was measured.

Transwell cell migration analysis. Cells (1×10⁵) of the drug-treated or vehicle control groups were seeded onto the top chambers of 24-well transwell plates (8 μm pore size; Corning Costar Biosciences, Acton, MA, USA). Hepatocyte growth factor (HGF, 20 ng/ml) (Biosource, Camarillo, CA, USA) was added to the bottom chambers as the chemoattractant. After incubation for 16 hours, cells in the top chamber (non-migrated) were removed by PBS rinsed cotton-swab; cells in the bottom chamber (migrated) were fixed by 4% paraformaldehyde and stained with 0.1% Giemsa solution (Sigma). The numbers of migrated cells were counted under a light microscope in 5 randomly-selected fields for each chamber.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was extracted from cells using Trizol® reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's recommendation. The first strand cDNA was synthesized with a random primer (N₆) using RT Beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Real-time quantitative PCR (qPCR) was performed using the ABI PRISM 7700. The DNA-intercalating SyBr green reagent (PE Applied Biosystems, Foster City, CA, USA) was used for detection of the PCR product. PCR amplification was subsequently performed with primers for E-cadherin, sense: 5′-TGA AGG TGA CAG AGC CTC TGG AT-3′ and antisense: 5′-TGG GTG AAT TCG GGC TTG TT-3′; for PPARγ, sense: 5′-TGC AGG TGA TCA AGA AGA CG-3′ and antisense: 5′-AGT GCA ACT GGA AGA AGG GA-3′; for β-actin, sense: 5′-TGG CAT TGC CGA CAG GAT-3′and antisense: 5′-GCT CAG GAG GAG CAA TGA TCT-3′. The expression level was estimated using the ΔΔCt method in which all data were normalized against the level of β-actin in each sample.

Statistical analyses. The means and standard deviations (SD) were calculated from at least three independent experiments. Student's t-test was used for comparing the means of two treatment groups. Multiple-group comparisons were carried out using one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test. Differences were considered statistically significant when p<0.05.

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Figure 1.

Effects of thiazolidinediones on E-cadherin expression, cell growth and motility of HepG2 cells. After treatment with different doses of rosiglitazone, troglitazone (from 1 μM to 100 μM), or dimethylformamide (DMF)-vehicle control (−) for 2 days, Western blot analysis was performed to measure the protein level of E-cadherin (a). SRB colorimetric assay showing the effects of different doses of rosiglitazone and troglitazone on the growth of HepG2 cells (b). Microphotographs of the migrated cells were taken at ×200 magnification with an Olympus optical microscope. Cell motility was determined using transwell chamber after cells were treated with 5 μM rosiglitazone (Ros. 5 μM) or 20 μM troglitazone (Tro. 20 μM) for 2 days (c). **p<0.01 when compared with vehicle control cells by Student's t-test.